HIV-1 latency model for high throughput screening

ABSTRACT

Isolated, latently infected T cell lines are provided that can be utilized in high throughput screening to discover compounds capable of activating HIV-I. The T cell lines harbor a latent HIV-I derived vector pro virus, which upon activation expresses a marker for late viral gene expression due to the insertion of the marker gene in the position of HIV-I envelope.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/742,241, filed Dec. 5, 2005, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This application relates to a novel HIV-1 latency model that can be usedfor high throughput screening to identify novel small molecules that canbe employed to eradicate latent virus from infected individuals.

BACKGROUND OF THE INVENTION

The advent of highly active antiretroviral therapy (HAART), whichinvolves the use of three or more antiretroviral drugs, has led to asignificant improvement in the care and survival of patients infectedwith HIV-1. In patients not infected with resistant strains of thevirus, HAART typically results in a dramatic decrease in viral loadoften from levels of 10,000-100,000 RNA copies/ml of plasma to less than50 copies/ml (3).

Given the dramatic effects of HAART, it was proposed that completeelimination of the virus might be possible within 2 to 3 years (36).However, even after long-term suppression of viral replication withHAART, the virus rapidly rebounds after therapy is discontinued (7,12).A key contributor to viral rebound appears to be a reservoir of latentlyinfected cells, including CD4⁺ memory T cells. The half-life of thelatently infected population is quite long, and it is estimated that itwould take over 60 years of HAART to eliminate this population (15).Therefore, life-long HAART would be required to control infection inpatients.

Retroviruses, including HIV-1, are RNA viruses that replicate through aDNA intermediate and integrate very efficiently into the genome of aninfected cell forming a provirus. Once the provirus is formed, it ismaintained in the genome of the infected cell and transferred todaughter cells in the same fashion as any other genetic element withinthe cellular genome. Thus, the virus has the potential to persist if itinfects long-lived cells such as memory T cells. It has been known since1986 (17) that HIV-1 can establish a latent infection in culture. It wasfound that a human T cell line infected with replication-competent viruscould develop a latent infection in which the provirus was dormant butcould be reactivated upon stimulation. Since then it has beenestablished that a number of cytokines including tumor necrosis factor(TNF)-α and even a small molecule such as the phorbol ester, phorbol12-myristate 13-acetate (PMA) can reactivate latent proviruses (30).

The role that latency is playing in preventing clearance of the virusinfection has become evident in recent years. Patients that had beensuccessfully treated with HAART in which viral RNA was maintained atlevels below 50 copies/ml in the plasma for years, experienced rapidvirus rebound upon withdrawal of therapy (7,12). Moreover, it was foundthat after T cell activation, virus could be isolated from CD4⁺ T cellstaken from these patients making it clear that to eradicate the virus itwill be necessary to eliminate the latently infected cells(10,16,19,45).

There have been attempts to flush the latent virus from infectedindividuals by non-specific activation of T cells to “turn on” latentproviruses. As part of this approach, the patients remain on HAART toprevent new infections, and the infected cells from which the latentproviruses are activated should die due to cytotoxic effects of viralexpression and/or because of targeting by the immune system which canrecognize the cells once they begin to express the viral proteins (3).One approach employed the combination of a monoclonal antibody againstCD3 on T cells plus IL-2 to activate T cells and consequently the latentproviruses (37). Other approaches have used IL-2 with or withoutadditional cytokines (8,13,31,41). To date, none of these protocols havebeen successful, and at least some of them have toxic side effects,which is not surprising considering the massive T cell activation thatoccurs. One plausible reason for the lack of latent provirus clearancecould be due to the inability of the therapeutic regime to reach all ofthe latent reservoirs.

A potentially fruitful approach to eliminating virus infection would beto identify small molecules with pharmacological properties that allowthese molecules to reach hard to access latent reservoirs in order toactivate latent proviruses. There is precedent for a small molecule thatcan activate latent HIV-1 proviruses, since it was found that thetumor-promoting phorbol ester PMA could stimulate latent virus. This hasled to recent studies with a non-tumor promoting phorbol ester,prostratin, which has also been found to be able to activate latentvirus leading to the hypothesis that prostratin can be employed to helperadicate latent infection (2729). However, it is not presently knownwhether prostratin has the appropriate pharmacological properties toenable total clearance of latent virus nor is it certain that only onedrug will be enough for latent virus elimination. Moreover, it wasrecently reported that prostratin displayed significant cytotoxicityputting into question its use in a clinical setting (43).

Thus, there is a need in the art for further strategies to discover newdrugs capable of activating latent HIV-1. Preferably, there is a needfor a cell-based assay that can be utilized in high throughput screening(HTS) to discover novel compounds capable of activating latent HIV-1.

SUMMARY OF THE INVENTION

The present invention solves a need in the art by providing an isolated,latently infected T cell line that can be utilized in high throughputscreening to discover compounds capable of activating HIV-1.

In particular, the present invention provides an isolated T cell lineharboring a latent HIV-1 derived vector provirus, which upon activationof the provirus expresses a secretable marker for late viral geneexpression, the gene for said marker being inserted in the position ofHIV-1 envelope.

Further provided is an in vitro cell-based method of identifyingcompounds capable of activating latent HIV-1. The method includesproviding an isolated T cell line harboring a latent HIV-1 derivedvector provirus, which upon activation of the provirus expresses asecretable marker for late viral gene expression due to the insertion ofthe marker gene in the position of HIV-1 envelope; and providing acandidate compound. The method further includes combining T cells fromthe T cell line with the candidate compound; and monitoring proviralactivation in the presence of the candidate compound as compared to inthe absence of the candidate compound to determine if the compound iscapable of activating latent HIV-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the HIV-I transducing vector and outline ofthe protocol for establishing latent clonal cell lines. (A) NLE⁻S-G is alentiviral vector that contains all the cis-acting elements required forreplication as well as two reporter genes: seap in the env position andegfp positioned 5′ to the start of codon of nef. Abbreviations: LTR,long terminal repeats; rre, Rev response element; ψ, packaging signal;seap, secreted alkaline phosphatase; egfp, enhanced green fluorescentprotein. Arrowheads represent partial deletions of viral sequence. (B)Vector virus was propagated in 293T cells by transient co-transfectionwith plasmids expressing pol and VSV-G-env (plasmids pCMVΔR8.2 andpMD.G, respectively). Most of the viral cis-acting elements in pCMVΔR8.2were deleted to prevent the likelihood of producingreplication-competent virus. Vector virus was then used to infect SupT1cells. Three or four weeks post-infection cell clones were isolated bylimiting dilution. Only clones producing low or undetectable levels ofseap that could be reactivated with 100 ng/ml TNF-α were used toestablish cell lines.

FIG. 2. Infection of SupT1 cells by NLE⁻S-G vector virus. (A) The upperpanels depict uninfected SupT1 cells. The lower panels show SupT1 cellsthat have been infected with NLE-S-G virus. The phase contrast (leftpanels) and fluorescence microscopy (right panels) images were capturedfive days post-infection. (B) Flow cytometric analysis of SupT1 cellseither mock infected or infected with NLE⁻S-G analyzed at day fivepost-infection or three weeks post-infection. Mock infection wasperformed using a lentiviral vector that does not carry seap or egfp.Three weeks post infection mass population of unsorted NLE⁻S-G infectedSupT1 cells (10⁶ cells/ml) were stimulated with TNF-α (100 ng/ml). Cellswere analyzed forty-eight hours after exposure. Numbers represent thepercentages of gfp positive cells. (C) SEAP activity in the conditionedmedia from NLE⁻S-G infected unsorted SupT1 cells (10⁶ cells/ml) fourdays, three weeks and one month post-infection. Three weekspost-infection, a sample (10⁶ cells/ml) of a mass population of NLE⁻S-Ginfected SupT1 cells was treated with TNF-α (100 ng/ml) and analyzedfour days post activation. RLU, relative light units.

FIG. 3. Reactivation of early and late viral gene expression in threeclonal cell lines by TNF-α. (A) 19ST1NLESG, 24ST1NLESG, and 29ST1NLESGcells (10⁶/ml) were exposed to TNF-α (50 ng/ml) and four days after gfpexpressing cells were analyzed by flow cytometry. The left panels showthe histograms of unstimulated cells, and the right panels represent thehistogram analysis of TNF-α (50 ng/ml) stimulated cells. Numbers showthe percentages of gfp positive cells. (B) SEAP in the cultured media ofuntreated and TNF-α (50 ng/ml) stimulated 19ST1NLESG, 24ST1NLESG,29ST1NLESG cells two and four days post plating. (C) Fold induction ofSEAP expression in 19ST1NLESG, 24ST1NLESG, and 29ST1NLESG cells (10⁶/ml)after stimulation with TNF-α (50 ng/ml). Readings were taken on thesecond and fourth days post activation. Results shown are the means andstandard deviations of one of three representative experiments.

FIG. 4. SEAP activation in 24ST1NLESG cells at different concentrationsof TNF-α. SEAP synthesis by 24ST1NLESG cells (10⁶/ml) four dayspost-activation with various concentrations (0.1 to 100 ng/ml) of TNF-α.The numbers above the bars show the fold induction of enzymatic activitycalculated as ratio between the average value of RLU per sample inducedcells compared to the mean RLU value of uninduced cells. RLU, relativelight units. Results shown are the means and standard deviations of oneof three representative experiments.

FIG. 5. HIV-1 Gag p24 protein production in 24ST1NLESG cells. HIV-1 Gagp24 protein was detected in the cultured media of untreated and TNF-α(50 ng/ml) treated 24ST1NLESG cells (10⁶/ml) two and four dayspost-plating. Results shown are the means and standard deviations of oneor two representative experiments.

FIG. 6. Real-time RT-PCR for relative quantification of HIV-1 mRNAlevels in 24ST1NLESG cells before and after activation from latency.Quantitative real-time RT-PCR was performed with RNA isolated from24ST1NLESG cells (10⁶/ml) following one and four days after stimulationwith TNF-α (50 ng/ml). cDNA was synthesized with polyT oligo, and thereactions were completed with primers to viral (Rev2) and cellular(β-actin) genes with SYBR green detection. The ‘Delta-delta method’ (PEApplied Biosystems) was used to compare the relative expression results(ΔC_(T)) between the values obtained from the RNA samples fromstimulated cells to the values obtained from RNA isolated from untreatedcells. The results were normalized to the expression of β-actin. Thedata are presented as the mean and standard deviation from twoexperiments.

FIG. 7. Southern analysis to confirm clonality and the structure of thevector provirus. Genomic DNA samples from 19ST1NLESG, 24ST1NLESG,29ST1NLESG cell clones, and parental SupT1 cells were digested with Xba1or EcoRV and subjected to Southern analysis. (A) Diagram of the viralvector NLE⁻S-G indicated the probe that is complimentary to the egfpgene and detects specific 8.8 kbp fragment from EcoRV digestedproviruses. The distance from XbaI restriction site to the end of 3′LTRis illustrated. (B) Southern blot showing the 8.8 kbp diagnostic bandresulting from EcoRV digestion in all three clones (lanes 7-9) and thefragments of various sizes generated by XbaI digestion due to thedifferent site of provirus integration in the host genome (lanes 2-4).Lanes 1 and 6 are positive controls (+) and were loaded with 50 pgplasmid DNA digested with XbaI and EcoRV, respectively. Genomic DNA fromparental cell line SupT1 was used as a carrier (lanes 1 and 6) or as anegative control (−) (lane 5).

FIG. 8. Z′ Factor Determination. 24ST1NLESG (10⁵/well) cells were seededin 96-well plates and both uninduced, (●) and induced (♦) cells wereassayed for SEAP activity after 48 h. Cells were treated with 50 ng/mlTNF-α (A), 1 mM valporic acid (VPA) (B), and 100 ng/ml PMA (C). Theresults from 88 samples per plate were compiled and the Z′ factor wasdetermined using the formula given in the section entitled assayreliability.

DETAILED DESCRIPTION OF THE INVENTION

As described above, highly active antiretroviral therapy (HAART) has hadan important impact upon morbidity and mortality from AIDS. AlthoughHAART results in a remarkable suppression of HIV-1 replication ininfected patients, it does not provide for elimination of the virus evenafter years of suppressive therapy. Complete viral clearance cannot beachieved due to the presence of latently infected cells in patients,which upon withdrawal of HAART, contribute to viral rebound. Attempts ateradicating latently infected cells by activating them with cytokinesand lymphokines has not met with success probably owing both to theinability of this treatment to reach all of the latent viral reservoirsand to the toxicity of the regimen. Small molecules with pharmacologicalproperties that allow them to reach all viral reservoirs and activatelatent HIV-1 proviruses could very well result in clearance of HIV-1infections when used in combination with HAART.

The present invention is directed, at least in part, to the developmentof a latently infected T cell line that can be used for high throughputscreening (HTS) to identify small molecules that can be employed toeradicate latent virus from infected individuals. In some embodiments,an isolated T cell line of the present invention harbors a latent HIV-1derived vector provirus, which upon activation expresses a secretableenzyme, such as secretable alkaline phosphatase (SEAP), as a marker forlate viral gene expression due to insertion of the marker gene in theposition of HIV-1 envelope.

In some embodiments, the secretable marker is capable of being detectedusing chemiluminescence. For example, SEAP production can be monitoredby employing a sensitive chemiluminescent reaction.

In some further embodiments, the T cell lines of the present inventionharbor a latent provirus capable of being activated by stimuli selectedfrom TNF-α, PMA, valporic acid and combinations thereof. For example,the isolated T cell line of the present invention harbors a latentprovirus that was activated using various stimuli previously shown toinduce latent HIV-1, including TNF-α, PMA, and valporic acid. Areproducible signal was detected in a small well format. The excellentreliability of the assay was characterized by a Z′ factor with valuesranging between 0.55 and 0.80.

In some embodiments, the latent provirus in the cell line isreplication-incompetent. For example, because the latent provirus wasderived from a defective HIV-1 vector, it allows HTS to be performedsafely since replication-competent virus would not be produced duringscreening. Thus, this system provides a safe, sensitive, and reliableassay for novel drug discovery aimed at eradication of HIV-1 infection.

In some embodiments, the T cell lines of the present invention harborlatent HIV-1 vector proviruses containing two marker genes that can beused for antiviral drug discovery, as well as for studying the mechanismof HIV-1 latency.

In some embodiments, seap marker makes the system amenable to highthroughput screening and given its placement in the genome reflectsactivation of viral late gene expression.

In some other embodiments, the latent provirus expresses a marker forviral early gene expression at the single cell level. For example, insome embodiments, a gene encoding a fluorescent protein, such as theegfp gene, allows monitoring activation of the viral early geneexpression at the single cell level. In addition, it provides adifferent marker in the system that can be used as a rapid secondaryscreen to control for artifacts that may arise when monitoring seapexpression, such as a particular compound yielding a false positive.Fluorescence from a fluorescent protein may be detected usingfluorescent microscopy, flow cytometry or combinations thereof, forexample.

Furthermore, in some embodiments, the latent provirus contains an intactHIV-1 gag gene, thereby allowing the use of Gag expression as a furthermarker of viral gene expression. For example, in some embodiments, Gagexpression may be used as an additional marker for rapid secondaryscreening of putative “hit” compounds.

Another important characteristic of the latency model described here isthat it yields excellent reliability in a small well format as reflectedby the Z′ scores obtained in assays with one of the cell lines, which isimportant for successful HTS. Moreover, since the vector provirus isreplication-incompetent, it provides a level of safety needed whenassaying a large number of samples as would be the case for HTS usingrobotic screening.

It is noteworthy that the cell lines have been cultured extensively forat least six months and still retain the same characteristics describedabove. This indicates that the cell lines are quite stable over time.

Despite the interesting research done on the mechanism of HIV-1 latency,there is still no clear understanding about how latency is establishedand maintained. It is apparent that latency is characterized by a stateof relative transcriptional inactivity. This is the case both in vivo inquiescent CD4⁺ memory T cells (22,32) and in vitro in previouslyestablished models of HIV-1 latency (30,39). Another common theme tolatency in vivo and in vitro is that various cytokines as well as otheractivators such as phorbol esters can activate latent virus(18,28,30,37,39,42). These attributes are reflected in the cell lines ofthe present invention in which gene expression is low until activatedwith the appropriate stimulus.

The in vitro cell-based model provided by the present inventionreproduces the major molecular characteristics of latency observed invivo. These similarities lend confidence that the in vitro cell model isuseful for identifying compounds that can activate latent proviruses.Candidate compounds identified using the cell-based methods of thepresent invention may be further tested for their ability tosuccessfully activate latent proviruses from patient samples.

The in vitro cell-based method of identifying compounds capable ofactivating latent HIV-1 includes providing an isolated T cell lineharboring a latent HIV-1 derived vector provirus, which upon activationof the provirus expresses a marker for late viral gene expression due tothe insertion of the marker gene in the position of HIV-1 envelope; andproviding a candidate compound. The method further includes combining Tcells from the T cell line with the candidate compound; and monitoringproviral activation in the presence of the candidate compound ascompared to in the absence of the candidate compound to determine if thecompound is capable of activating latent HIV-1.

In some embodiments, the monitoring step further includes combining Tcells from the T cell line with a positive control for latent proviralactivation; and monitoring proviral activation in the presence of thepositive control. For example, the positive control may be a stimuliselected from, but not limited to, the following: tumor necrosis factor(TNF)-α, phorbol 12-myristate 13-acetate (PMA), valporic acid andcombinations thereof. In some further embodiments, the method ofidentifying compounds capable of activating latent HIV-1 may furtherinclude determining the ability of a candidate compound to activatelatent HIV-1 from patient samples.

The T cell line used in the method of the present invention harbors alatent provirus. The latent provirus contains a gene encoding asecretable marker for late viral gene expression. Upon activation of thelatent provirus, the T cells secrete the marker for late viral geneexpression. For example, the secretable marker may be a secretableenzyme, such as secretable alkaline phosphatase. In this instance, themonitoring step in the method may include detecting enzymatic activityof the secreted alkaline phosphatase in the presence of an alkalinephosphatase substrate. Examples of suitable chemiluminescent substratesfor alkaline phosphatase include CSPD® and CDP-Star®, which areavailable from Applied Biosystems (Bedford, Mass.). However, the presentinvention is not limited to these.

The latent provirus in the cells used in the method of the presentinvention may further contain a marker gene for early viral geneexpression. In some embodiments, the expressed marker for early viralgene expression is a fluorescent protein, such as a green fluorescentprotein. In this instance, the method of the present invention mayfurther include detecting cell fluorescence by fluorescent microscopy,flow cytometry or combinations thereof.

The latent provirus in the cells employed in the method of the presentinvention preferably contain an intact HIV-1 Gag gene. This allows theuse of Gag expression by the cells as an additional marker for rapidsecondary screening of putative “hit” compounds. Therefore, in someembodiments, the inventive method includes detecting HIV-1 Gagexpression.

Although the background levels of gene expression are low for the celllines of the present invention, some expression is still detectable evenfor the 24ST1NLESG line with the lowest background levels of expression(FIG. 3). This has also been noted in other in vitro models of latency(30,39) and it has also been observed in vivo. For example, when viralmRNA levels are examined in highly purified CD4⁺ memory cells frompatients successfully undergoing HAART suppressive therapy, low butdetectable levels of viral RNA can be found in the absence of virusproduction (32). Once activated these cells are capable of producingvirus (5,9,19,24). Thus, it is not unexpected that some backgroundexpression from the latent provirus would be found in the model systemof the present invention. Moreover, the background expression is not thesame for the three cell lines depicted in FIG. 3.

One potential influence upon the background level of expression is thesite of integration. Since the proviruses are integrated in differentpositions in the genome (FIG. 5), the local site of integration caninfluence expression from the provirus and could, at least in part,account for the different levels of background expression (23,26,44).

Another characteristic of the cell clones described here was theapparent variegated expression of egfp within each clonal population(FIG. 3A). Upon activation with TNF-α, the number of cells expressingegfp was quite high ranging from 82% to 90% of the cells by day 4post-activation. However, a relatively small fraction of the cells werenot activated (FIG. 3A). This variegated expression indicates that theremight be some level of epigenetic regulation of expression. This mightbe related to disparate responses of cells to TNF-α stimulation indifferent phases of the cell cycle (39) or due to heterochromaticpackaging (14). This suggests that it may be necessary to administer atherapeutic activator repeatedly to patients or that a cocktail ofactivators may be required to “flush” the virus from all of its latentreservoirs.

As described herein, additional cell clones were isolated with higherlevels of background expression, yet with excellent levels ofinducibility. Initial screening for compounds that activate latent viruswould be done employing the 24ST1NLESG cell line given its lowbackground and high degree of inducibility. However, the other cellclones can be utilized for secondary screening. Any promising compoundidentified by HTS would be anticipated to also be able to activatelatent proviruses integrated in different locations. The other cellclones provide a rapid and safe secondary screen that would be able toquickly rule out artifacts such as effects dependent upon the specificsite of integration.

The present invention provides the first HIV-1 latency model that hasbeen developed that can be used for high throughput screening toidentify novel small molecules that can be employed to eradicate latentvirus from infected individuals.

Vector System

The present inventors have developed a cell-based assay that can be usedin HTS to discover novel compounds that can activate latent HIV-1proviruses. The marker gene that is being utilized in this system is thesecretable alkaline phosphatase (seap) gene, a truncated form ofplacental alkaline phosphatase lacking the membrane anchor so that it issecreted (2). It provides a very sensitive chemiluminescent assay whenused in conjunction with the alkaline phosphatase substrate CSPD and assuch is amenable to HTS. It has been shown that as little as ⁻¹³ g ofplacental alkaline phosphatase can be detected (4,46). Other advantagesare (2,46): (1) SEAP is heat stable and resistant to the alkalinephosphatase inhibitor L-homoarginine, which allows endogenous alkalinephosphatase (AP) activity to be eliminated via pretreatment at 65° C.with the inhibitor; (2) because it is secreted, there is no necessityfor preparation of cell lysates; (3) the chemiluminescence assay has alinear dynamic range of over four orders of magnitude, which helpscontrol experimental parameters to obtain readouts in the linear rangeallowing valid comparisons among the different samples, and (4) thesignal reaches a maximum at approximately 10 minutes after the reactionand remains stable for at least an hour which augments thereproducibility of the assay.

Since a hallmark of active replication is production of the HIV-1 lateproteins including the viral envelope polyprotein (Env), the seap genewas inserted in the env position to serve as an indicator of late geneexpression (FIG. 1). Disruption of the env gene by insertion of seap inplace of the env start codon, imparts a level of safety to the system bypreventing production of the essential Env polyprotein. However, inorder to include an additional level of safety to prevent production ofreplication-competent virus, 2.5 kbp of the pol gene was also deleted,since it has not been reported to contribute to the establishment oflatency but is essential for replication (FIG. 1).

In addition to the seap gene, the enhanced green fluorescent protein(egfp) reporter gene was inserted 5′ to the nef start codon such that itshould be expressed from the multiply spliced nef mRNA (FIG. 1) (38,40).The egfp gene was included in the system to provide a marker that wouldallow monitoring of infection by fluorescent microscopy and flowcytometry as well as providing a marker for subsequent isolation oflatently infected cell clones.

Vector Characterization

In order to propagate vector virus, the vector was cotransfected into293T cells along with plasmids pCMVΔR 8.2 and pMD.G (33) to complementthe pol and env defects, respectively. It is noteworthy that pMD.Gexpresses the vesicular stomatitis G protein which allows the productionof pseudotyped vector virus that can be readily concentrated byultracentrifugation (6,35). Vector virus supernatants were collected,concentrated, and used to infect the human lymphocyte based cell lineSupT1 via spinoculation (Examples) (34). A surprising observation wasmade when SupT1 cells were transduced with the vector pNLE S-Gcontaining an intact vpu start codon. Although vector virus couldeffectively transduce the SupT1 cells as evidenced by EGFP expression inthe infected cells, SEAP was not expressed (data not shown). HIV-1 envis translated from a vpu-env bicistronic mRNA (20,38). It was believedthat translation initiation of env occurs due to a leaky scanningmechanism (20). It was possible that due to the insertion of SEAP, leakyscanning was inhibited. To address this problem, the start codon of vpuwas destroyed to allow SEAP expression from a monocistronic RNA. Theresultant vector virus could efficiently infect SupT1 cells as well asexpress significant levels of SEAP after 4 days post-infection (FIG. 2).

The infected mass population of cells was next tested for its ability toestablish a latent infection. The infected mass population of SupT1cells was maintained in culture for one month. By week three, flowcytometry indicated that most of the infected cells had died and/orreverted to a latent phenotype as denoted by the reduction of the GFPpositive cells declining from 63% on day 5 to 2% 3 weeks post-infection(FIG. 2B). This was also reflected in the reduction of SEAP expressionby the third week (FIG. 2C). It was shown in previous studies that tumornecrosis factor (TNF)-α could activate HIV-1 virus production fromlatently infected cell lines (30). The mass population was treated withTNF-α, and it was found that there was a significant increase in thenumber of GFP-positive cells to 19% as well as a 400-fold increase inSEAP activity (FIGS. 2B and C). These results clearly indicated that thevector could form a latent infection and activation could be monitoredby SEAP expression.

Isolation and Characterization of Latently Infected Cell Clones

In order to provide a well characterized system to identify moleculesthat can activate latent virus, SupT1 cell clones harboring latentvector provirus were isolated. Cell clones were isolated by limitingdilution according to the standard protocol (11). Clones were thentested to determine if they harbored latent virus that could bestimulated with TNF-α. Flow cytometry to monitor GFP expression showedthat after treatment with TNF-α there was a significant increase in thenumber of cells expressing GFP within each of the three clonalpopulations (FIG. 3A). All three lines also exhibited a significant foldinduction in SEAP activity after treatment with TNF-α. By day 2 posttreatment, SEAP activity increased by approximately 50-, 400-, and150-fold in lines 19ST1NLESG, 24ST1NLESG, 29ST1NLESG, respectively(FIGS. 3B and C). The variation in the fold induction of the three cellclones was primarily due to differences between the background levels ofexpression (FIG. 3B). Given the relatively low background for24ST1NLESG, and its high level of induction in SEAP activity reachingapproximately 600-fold by day 4, attention was focused upon this cellline. To test the sensitivity and concentration dependence of theinducible 24ST1NLESG cell line, it was stimulated with variousconcentrations of TNF-α. Even at 0.1 ng/ml of TNF-α induction could bedetected yielding SEAP activity 4-fold above background (FIG. 4).

Besides the two exogenous marker genes, seap and egfp, autologous HIV-1gag gene expression can also be used as a marker for activation sincethe gag gene remains intact in the system. The Gag polyprotein istranslated from full-length viral RNA, which is expressed during thelate stage of viral infection. Monitoring of p24^(gag), the viral capsidprotein, could be useful for a secondary screen since there is asensitive and straightforward ELISA available to measure its expression.

To further characterize the 24ST1NLESG cell line, p24^(gag) expressionwas monitored using the ELISA after treatment with TNF-α (50 ng/ml)(FIG. 5). As anticipated, the concentration of p24^(gag) increasedsignificantly at 2 and 4 days post-activation reaching levels 50 foldand 100-fold above background, respectively (FIG. 5).

The consistent reactivation of viral genes as evidenced by the patternof expression of the three markers assayed correlated with an increasein the relative levels of total viral mRNA. The relative amount of totalHIV-1 mRNA was determined by quantitative real-time PCR using primers tothe second exon of rev (Rev2). Rev2 primers were chosen to amplify theviral transcripts since they are complimentary to sequences presented inall RNA species encoded by the provirus (38). RT PCR was primed witholigo d(T)16. Therefore, only the level of mature polyadenylated RNA wasdetected.

The relative ratio of viral RNA after stimulation of 24ST1NLESG latentcells was determined as described in the Examples using the relative RNAresults obtained for the unstimulated 24ST1NLESG cells as a control.β-actin was used as a reference gene. By day 4 post-stimulation withTNF-α (50 ng/ml), the relative ratio of viral RNA increased by at least100-fold indicating that the increase in proviral protein expressionresulted due to a significant increase in the steady-state levels ofviral RNA (FIG. 6).

Southern blotting was performed to examine the proviral structure andthe clonal nature of the cell lines. Genomic DNA was isolated from eachof the cell clones followed by digestion with the EcoRV or XbaI andsubsequent Southern analysis probing with egfp-specific sequence (FIG.7). EcoRV cuts once within each LTR and would be expected to yield an8.8 kbp band from each sample, which was the case (FIG. 7). XbaI cutsonce within the provirus. Therefore, if each line represents a differentcell clone, XbaI would be anticipated to yield a different size bandbecause, given the random nature of proviral integration, the XbaI sitein the adjacent genomic DNA will be in a different location. The XbaIdigestion yielded bands of different size, between 5 and 6 kbp,indicating the three lines are different clones with disparate proviralintegration sites (FIG. 7).

An advantage to using a defective HIV-1 provirus for monitoring proviralactivation is that replication-competent virus is not produced, which isparticularly important for HTS when a large number of samples areanalyzed. However, it is prudent to test before wide-scale use of thesystem that RC virus was not produced during the development of the celllines. To that end, the cell lines were analyzed for reversetranscriptase activity after treatment with 50 ng/ml of TNF-α. Sincemost of the pol gene was deleted from the vector, the cell lines shouldbe negative for RT activity, which was the case (data not shown).Without RT activity, it should not be possible to passage RC virus.Nevertheless, the cell lines were tested further. As shown in FIG. 5,24ST1NLESG cells produce HIV-1 capsid proteins. To prove that onlydefective virions were formed, an HIV-1 gag transfer assay was performedas described in the Examples. This test confirmed the lack of RC virus.

Assay Reliability

For the assay to be useful in a HTS it should be reliable in a smallwell format. Assay optimization and validation requires thedetermination of the Z′ factor, a dimensionless statisticalcharacteristic used to assess the quality of data generated in apotential HTS assay (48). It is a commonly used measure of assayperformance and reliability that takes into account both the assaysignal dynamic range (signal-to-background) and variation(signal-to-noise) associated with the measured signals.

The Z′ factor values for the latency assay of the present invention werecalculated based on the analysis of SEAP expression from uninduced(negative) and induced (positive) 24ST1NLESG cells. Eighty-eight wellsof a 96-well plate were seeded with 10⁵ cells/well either in media aloneor media containing an inducer in a final volume of 20 μl. The remaining8 wells contained serial dilutions of SEAP protein as a positive controlfor the assay. The plates were assayed for the presence of SEAP after 48hours. The readouts for each plate were compiled and the Z′ factor foreach inducer and the uninduced control was determined using theequation:

$Z^{\prime} = {1 - \frac{\left( {{3\;\sigma_{positive}} + {3\;\sigma_{negative}}} \right)}{{\mu_{positive} - \mu_{negative}}}}$where σ represents the standard deviation and μ is the mean of each setof data points. A perfect assay would have a Z′ factor value of 1, whilean excellent assay would score between 0.5 and 1. If the Z′ value fallsbetween 0 and 0.5, the performance of the assay should be improved. Anyscore≦0 is indicative of an insignificant separation between thebackground and the positive signal and the assay must therefore beredesigned (47). Since the Z′ factor is dimensionless, the reliabilityof assays performed separately but which are similar in design may bedirectly compared.

The Z′ factors for the latency assay with 24ST1NLESG cells of thepresent invention are shown in FIG. 8. Induction with 50 ng/ml TNF-α ina 96-well format resulted in a Z′ factor of 0.8, while activation with 1mM VPA and 100 ng/ml PMA returned Z′ scores of 0.63 and 0.55,respectively. A certain amount of variability in these assays wasexpected since pipetting by hand during seeding and performance of theSEAP detection assay can introduce some variation. Even with thisinherent variability, the Z′ values for all three tested compounds areabove 0.5, indicating excellent assay performance.

EXAMPLES Example 1 Packaging and Transducing Vectors

The recombinant transducing vector used in this study was based on theNL4-3 hemigenomic plasmid. A 2.5 kb deletion in the pol gene was made bysplicing by overlap extension (25). Three PCR products were generated:(i) sequence between the unique BssHII site (coordinate 710) and thestop codon of gag at 2300, (ii) sequence stretching from 100 bp upstreamof the start codon of vif (coordinate 4840) to the unique EcoRI site(coordinate 5744), (iii) a 2.5 kb product formed by linking products iand ii by PCR sequence overlap extension (SOE). The PCR product (iii)containing the deleted pol gene was cloned into pGEM (Promega) and wasexcised from there with BssHII and EcoRI followed by ligation into theBssHII/EcoRI digested NL4-3 backbone yielding pNL2.5p⁻. The same methodwas used to insert the egfp coding sequence 10 bp upstream from thestart codon of nef while deleting 80 bp from the 3′ of env: coordinates8700 to 8780. Five PCR products also were made: (a) from the uniqueBamHI site (coordinate 8465) in env to 50 bp downstream of the stopcodon of rev (coordinate 8680), (b) the egfp gene including the Kozaksequence (30), (c) products a and b linked by SOE PCR, (d) from thestart codon of nef to the unique XhoI site in nef, (e) products c and dlinked by SOE PCR. Product e was cloned into pGEM and cut from therewith BamHI and XhoI. The BamHI-XhoI fragment was ligated into thepNL2.5p⁻ backbone, which was digested with BamHI and XhoI to obtain theNLp⁻e⁺G construct.

The start codons of vpu and env were altered by site-directedmutagenesis (Stratagene). The NL4-3 genome from EcoRI to NheI (1.5 bp)was subcloned into a modified pSEAP-Basic vector, where BamHI-BamHIdeletion of the SEAP gene was made, and the ClaI restriction site wascreated in place of the start codon of env (e*).

The SEAP open reading frame was cut from pSEAP-Basic (CLONTECHLaboratories) with ClaI and BsmI and ligated into plasmid NLp⁻e*Glinearized with ClaI. The in-frame orientation of SEAP was confirmedwith restriction enzyme digestion and DNA sequencing. Thus pNLE⁻S-G(FIG. 1A) was created.

pCMVΔR8.2, a CMV promoter-driven HIV packaging plasmid with allaccessory proteins, was used to compensate for the lack of pol geneexpression. pMD.G was used to provide expression of vesicular stomatitisvirus G env (VSV-G)(33).

Example 2 Cell Culture and Reagents

293T cells were grown in minimal essential medium (MEM) supplementedwith 10% fetal bovine serum (Hyclone), 0.2 mM MEM non-essential aminoacid solution (GIBCO BRL 11095-080), 250 U/ml penicillin, and 250 μg/mlstreptomycin (GIBCO BRL 11140-050). SupT1 cells as well as 19ST1NLESG,24ST1NLESG, and 29ST1NLESG cells were maintained in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and 2 mM L-glutamine. TNF-α wasobtained from R&D Systems (Minneapolis, Minn.). PMA, and valporic acid,were purchased from Sigma (St. Louis, Mo.).

Example 3 Virus Production, Infection and Isolation of Latent Clones

Vector virus was produced using transient, three-plasmid co-transfectionvia modified calcium phosphate precipitation method (21). Twenty-fourhours after plating, 293T cells (2.5×10⁶ per 100-mm-diameter dish) wereco-transfected with pNLE⁻S-G (5 μg), pCMVΔR8.2 (5 μg), and pMD.G (4 μg).Viral supernatant was collected from 20 plates 48 h post-transfectionand was concentrated by ultracentrifugation for 1.5 h at 50,000 g in aBeckman 45 Ti rotor at 4° C. Viral particles were resuspended in 2 mlmedia with 8 μg/ml polybrene and the viral concentrate was used toinfect 5×10⁶ SupT1 cells. The inoculation was preceded by spinoculation(34) at 1,200 g for 2 h at 25° C. After 6 h of incubation at 37° C. and5% CO2, SupT1 cells were washed twice with PBS and resuspended in freshmedia. Four weeks post-infection, the mass population of SupT1 infectedcells was used to isolate clones harboring latent provirus. This wasdone by limiting dilution following the standard protocol (11). Only theclones that showed significant increase of SEAP activity afteractivation with 100 ng/ml TNF-α were chosen.

Example 4 Assays for Replication-Competent Virus

HIV gag transfer assay. 1×10⁶ SupT1 infected cells (latent masspopulation and clonal cell lines) were plated followed by harvestingsupernatant one week later. Samples were collected both from TNF-αactivated and untreated cells. They were passed through a 0.45-μmpore-size filter and used to inoculate 2×10⁶ parental SupT1 cells for 24h. The cells were washed twice with PBS and resuspended in fresh media.The same process was repeated once again with conditioned media from thetargeted parental cells, and a new batch of SupT1 cells was treated.Passing the conditioned media over two sets of virgin SupT1 cells helpsto assure that the carryover of gag protein in the samples will beeliminated and only the de novo synthesized protein, if present, will beassessed. Samples harvested after one week post-incubation were used todetermine the concentration of p24 using an HIV-1 p24^(gag) ELISA kitaccording to the manufacturer's protocol (Perkin Elmer Life Sciences).Samples were considered virus free when the p24^(gag) concentration wasbelow the detection threshold, which for this assay, was determined tobe 10 pg/ml.

Reverse transcriptase assay. Four days after plating the conditionedmedia from the clonal latent cell lines (both TNF-α stimulated anduntreated) was used to determine the activity of HIV-1 RT using acalorimetric enzyme immunoassay (Roche Applied Sciences). Thesensitivity threshold for this assay is 1 pg per reaction or 50 pg/ml.The detection limit according to the manufacturer's protocol was definedto be a signal level of twice the background, which in our assay wasrepresented by conditioned media from untreated parental SupT1 cells.

Example 5 Flow Cytometric Analysis

Flow cytometric analysis was performed with a FACScan (Beckman-Coulter)using CellQuest software. Prior to the flow cytometric analysis 1×10⁶cells were washed twice and resuspended in 1 ml of PBS.

Example 6 Southern Blotting

Isolation of genomic DNA and Southern blotting analysis were doneaccording to standard procedures (1). Genomic DNA (20 μg) was digestedwith EcoRV or XbaI and electrophoresed on a 0.8% agarose gel. Blots werethen hybridized with ³²P-labeled probe complimentary to egfp sequence inthe pNLE⁻S-G transducing vector.

Example 7 SEAP Assay for Detection of Late Gene Expression

1×10⁶ or 1×10⁵ cells from the clonal cell lines or mass populations ofinfected cells were resuspended in RPMI media with or without theactivator. At the indicated day post plating, samples were taken todetermine the activity of SEAP. The assay was performed using a SEAPDetection kit (BD Biosciences, Palo Alto, Calif.) according to themanufacturer's protocol. The relative light units (RLU) were measuredwith a tube luminometer (Turner Designs 20/20) or a MLX Microtiter PlateLuminometer (DYNEX Technologies).

Example 8 Quantitative Real-Time RT-PCR

Total cellular RNA was isolated from untreated or TNF-α (50 ng/ml)stimulated 24ST1NLESG (1×10⁶) cells using the RNeasy Mini Kit accordingto the manufacturer's instructions (QIAGEN, Maryland, USA). ExtractedRNA was subsequently treated with RQ1 DNase Kit (Promega, Madison Wis.,USA) to remove the traces of DNA. The removal of DNA was confirmed withreal-time PCR as described below by the lack of detectable signal abovebackground amplification observed in the no-template reactions. Two-stepreal-time PCR was performed for relative quantification of viral RNA.cDNA was synthesized with TaqMan reverse transcription reagents andoligo d(T)16 following the protocol supplied by Applied Biosystems(Roche N.J., USA).

For each experiment, cDNA was synthesized with 5 μg of DNase treated RNAin 50 μl reaction volumes incubated at 25° C., 10 min; 48° C., 30 min,and 95° C., 5 min. Real-time PCR was accomplished with SYBR Green PCRMaster Mix and run in DNA Engine Opticon 2 (MJ Research) detector withOpticon Monitor Analysis software version 1.4. Reactions received 5 μlof cDNA and 2.5 μM of each primer in a 25 μl reaction volume. The primerpair sequences used in the reactions are as follows:

forward 5′-CTGGAACGGTGAAGGTGACA-3′ (SEQ ID NO:1), and

reverse 5′-AAGGGACTTCCTGTAACAATGCA-3′ (SEQ ID NO:2) for β-actin;

forward 5′-AGGTGGAGAGAGAGACAGAGACA-3′ (SEQ ID NO:3), and

reverse 5′-TCCCAGAAGTTCCACAATCC-3′ (SEQ ID NO:4) for Rev2. Real-time PCRwas carried out with a single thermocycle protocol of 95° C., 3 min; and41 cycles of 95° C., 20 s, followed by 55° C., 1 min.

For each primer set, amplification efficiencies were determined byobtaining a standard curve with serial dilutions of cDNA from stimulatedcells; the log of the relative target quantity was plotted against theCT (cycle threshold) value. The standard curves with slopes −5.53 and−5.13 showed amplification efficiencies 98 and 99% for β-actin and Rev2primer pairs, respectively. A dissociation curve was generated for eachprimer pair and demonstrated the amplification of a single product. Thesizes of the amplified products were confirmed by agarose gelelectrophoresis and the specificity of the products was confirmed bysequencing. Reactions were completed in duplicate and no-templateno-reverse transcriptase controls were included per primer pair.Relative RNA abundance ΔCT per sample was determined as the differencebetween the target gene CT value and the CT observed for the referencegene. The relative expression ratio was calculated using the formula2^(−ΔΔCt) (‘Delta-delta method’ PE Applied Biosystems), where ΔΔCt isthe difference between the relative RNA abundance in the sample inquestion and the control sample. The ΔCT of the untreated 24ST1NLESGcells was used as a control to the relative RNA abundance in TNF-αstimulated cells.

REFERENCES

-   1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.    Seidman, J. A. Smith, and K. Struhl. 1994. Current Protocols in    Molecular Biology. John Wiley & Sons, Inc., Hoboken, N.J.-   2. Berger, J., J. Hauber, R. Hauber, R. Geiger, and B. R.    Cullen. 1988. Secreted placental alkaline phosphatase: a powerful    new quantitative indicator of gene expression in eukaryotic cells.    Gene 66: 1-10.-   3. Blankson, J. N., D. Persaud, and R. F. Siliciano. 2002. The    challenge of viral reservoirs in HIV-1 infection. Annu. Rev. Med.    53:557-593.-   4. Brinkman, K., J. A. Smeitink, J. A. Romijn, and P. Reiss. 1999.    Mitochondrial toxicity induced by nucleoside-analogue    reverse-transcriptase inhibitors is a key factor in the pathogenesis    of antiretroviral-therapy-related lipodystrophy. Lancet    354:1112-1115.-   5. Brooks, D. G., P. A. Arlen, L. Gao, C. M. Kitchen, and J. A.    Zack. 2003. Identification of T cell-signaling pathways that    stimulate latent HIV in primary cells. Proc. Natl. Acad. Sci. U.S.A    100:12955-12960.-   6. Burns, J. C., T. Friedmann, W. Driever, M. Burrascano, and J. K.    Yee. 1993. Vesicular stomatitis virus G glycoprotein pseudotyped    retroviral vectors: concentration to very high titer and efficient    gene transfer into mammalian and nonmammalian cells. Proc Natl Acad    Sci USA 90:8033-8037.-   7. Chun, T. W., R. T. Davey, Jr., M. Ostrowski, J. J. Shawn, D.    Engel, J. I. Mullins, and A. S. Fauci. 2000. Relationship between    pre-existing viral reservoirs and the re-emergence of plasma viremia    after discontinuation of highly active anti-retroviral therapy. Nat.    Med. 6:757-761.-   8. Chun, T. W., D. Engel, S. B. Mizell, C. W. Hallahan, M.    Fischette, S. Park, R. T. Davey, Jr., M. Dybul, J. A. Kovacs, J. A.    Metcalf, J. M. Mican, M. M. Berrey, L. Corey, H. C. Lane, and A. S.    Fauci. 1999. Effect of interleukin-2 on the pool of latently    infected, resting CD4+ T cells in HIV-1-infected patients receiving    highly active anti-retroviral therapy. Nat. Med. 5:651-655.-   9. Chun, T. W., J. S. Justement, R. A. Lempicki, J. Yang, G. Dennis,    Jr., C. W. Hallahan, C. Sanford, P. Pandya, S. Liu, M.    McLaughlin, L. A. Ehler, S. Moir, and A. S. Fauci. 2003. Gene    expression and viral production in latently infected, resting CD4+ T    cells in viremic versus aviremic HIV-infected individuals. Proc.    Natl. Acad. Sci. U.S.A 100:1908-1913.-   10. Chun, T. W., L. Stuyver, S. B. Mizell, L. A. Ehler, J. A.    Mican, M. Baseler, A. L. Lloyd, M. A. Nowak, and A. S. Fauci. 1997.    Presence of an inducible HIV-1 latent reservoir during highly active    antiretroviral therapy. Proc. Natl. Acad. Sci. U.S.A 94:13193-13197.-   11. Coligan, J., A. Kruisbeek, D. Margulies, E. Shevach, and    Strober. W. 1991. Current Protocols in Immunology. Green Publishing    Associates and Wiley-Enterscience.-   12. Davey, R. T., Jr., N. Bhat, C. Yoder, T. W. Chun, J. A.    Metcalf, R. Dewar, V. Natarajan, R. A. Lempicki, J. W.    Adelsberger, K. D. Miller, J. A. Kovacs, M. A. Polis, R. E.    Walker, J. Falloon, H. Masur, D. Gee, M. Baseler, D. S.    Dimitrov, A. S. Fauci, and H. C. Lane. 1999. HIV-1 and T cell    dynamics after interruption of highly active antiretroviral therapy    (HAART) in patients with a history of sustained viral suppression.    Proc. Natl. Acad. Sci. U.S.A 96:15109-15114.-   13. Dybul, M., B. Hidalgo, T. W. Chun, M. Belson, S. A.    Migueles, J. S. Justement, B. Herpin, C. Perry, C. W.    Hallahan, R. T. Davey, J. A. Metcalf, M. Connors, and A. S.    Fauci. 2002. Pilot study of the effects of intermittent    interleukin-2 on human immunodeficiency virus (HIV)-specific immune    responses in patients treated during recently acquired HIV    infection. J. Infect. Dis. 185:61-68.-   14. Elgin, S. C. and S. I. Grewal. 2003. Heterochromatin: silence is    golden. Curr. Biol. 13:R895-R898.-   15. Finzi, D., J. Blankson, J. D. Siliciano, J. B. Margolick, K.    Chadwick, T. Pierson, K. Smith, J. Lisziewicz, F. Lori, C.    Flexner, T. C. Quinn, R. E. Chaisson, E. Rosenberg, B. Walker, S.    Gange, J. Gallant, and R. F. Siliciano. 1999. Latent infection of    CD4+ T cells provides a mechanism for lifelong persistence of HIV-1,    even in patients on effective combination therapy. Nat. Med.    5:512-517.-   16. Finzi, D., M. Hermankova, T. Pierson, L. M. Carruth, C.    Buck, R. E. Chaisson, T. C. Quinn, K. Chadwick, J. Margolick, R.    Brookmeyer, J. Gallant, M. Markowitz, D. D. Ho, D. D. Richman,    and R. F. Siliciano. 1997. Identification of a reservoir for HIV-1    in patients on highly active antiretroviral therapy. Science    278:1295-1300.-   17. Folks, T., D. M. Powell, M. M. Lightfoote, S. Benn, M. A.    Martin, and A. S. Fauci. 1986. Induction of HTLV-III/LAV from a    nonvirus-producing T-cell line: implications for latency. Science    231:600-602.-   18. Folks, T. M., K. A. Clouse, J. Justement, A. Rabson, E.    Duh, J. H. Kehrl, and A. S. Fauci. 1989. Tumor necrosis factor alpha    induces expression of human immunodeficiency virus in a chronically    infected T-cell clone. Proc. Natl. Acad. Sci. U.S.A 86:2365-2368.-   19. Fondere, J. M., G. Petitjean, M. F. Huguet, S. L. Salhi, V.    Baillat, A. ura-Biegun, P. Becquart, J. Reynes, and J. P.    Vendrell. 2004. Human immunodeficiency virus type 1 (HIV-1) antigen    secretion by latently infected resting CD4+ T lymphocytes from    HIV-1-infected individuals. J. Virol. 78:10536-10542.-   20. Furtado, M. R., R. Balachandran, P. Gupta, and S. M.    Wolinsky. 1991. Analysis of alternatively spliced human    immunodeficiency virus type-1 mRNA species, one of which encodes a    novel tat-env fusion protein. Virology 185:258-270.-   21. Gorman, C. 1985. High efficiency gene transfer into mammalian    cells, p. 143-190. In: D. M. Glover (ed.), DNA Cloning. IRL Press,    Oxford.-   22. Han, Y., K. Lassen, D. Monie, A. R. Sedaghat, S. Shimoji, X.    Liu, T. C. Pierson, J. B. Margolick, R. F. Siliciano, and J. D.    Siliciano. 2004. Resting CD4+ T cells from human immunodeficiency    virus type 1 (HIV-1)-infected individuals carry integrated HIV-1    genomes within actively transcribed host genes. J. Virol.    78:6122-6133.-   23. He, G. and D. M. Margolis. 2002. Counterregulation of chromatin    deacetylation and histone deacetylase occupancy at the integrated    promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1    repressor YY1 and HIV-1 activator Tat. Mol. Cell Biol. 22:2965-2973.-   24. Hermankova, M., J. D. Siliciano, Y. Zhou, D. Monie, K.    Chadwick, J. B. Margolick, T. C. Quinn, and R. F. Siliciano. 2003.    Analysis of human immunodeficiency virus type 1 gene expression in    latently infected resting CD4+ T lymphocytes in vivo. J Virol    77:7383-7392.-   25. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R.    Pease. 1989. Site-directed mutagenesis by overlap extension using    the polymerase chain reaction. Gene 77:51-59.-   26. Jordan, A., P. Defechereux, and E. Verdin. 2001. The site of    HIV-1 integration in the human genome determines basal    transcriptional activity and response to Tat transactivation.    EMBO J. 20:1726-1738.-   27. Korin, Y. D., D. G. Brooks, S. Brown, A. Korotzer, and J. A.    Zack. 2002. Effects of prostratin on T-cell activation and human    immunodeficiency virus latency. J Virol 76:8118-8123.-   28. Kulkosky, J., D. M. Culnan, J. Roman, G. Dornadula, M.    Schnell, M. R. Boyd, and R. J. Pomerantz. 2001. Prostratin:    activation of latent HIV-1 expression suggests a potential inductive    adjuvant therapy for HAART. Blood 98:3006-3015.-   29. Kulkosky, J., J. Sullivan, Y. Xu, E. Souder, D. H. Hamer,    and R. J. Pomerantz. 2004. Expression of latent HAART-persistent HIV    type 1 induced by novel cellular activating agents. AIDS Res. Hum.    Retroviruses 20:497-505.-   30. Kutsch, O., E. N. Benveniste, G. M. Shaw, and D. N. Levy. 2002.    Direct and quantitative single-cell analysis of human    immunodeficiency virus type 1 reactivation from latency. J Virol    76:8776-8786.-   31. Lafeuillade, A., C. Poggi, S. Chadapaud, G. Hittinger, M.    Chouraqui, M. Pisapia, and E. Delbeke. 2001. Pilot study of a    combination of highly active antiretroviral therapy and cytokines to    induce HIV-1 remission. J. Acquir. Immune. Defic. Syndr. 26:44-55.-   32. Lassen, K. G., J. R. Bailey, and R. F. Siliciano. 2004. Analysis    of human immunodeficiency virus type 1 transcriptional elongation in    resting CD4+ T cells in vivo. J. Virol. 78:9105-9114.-   33. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H.    Gage, I. M. Verma, and D. Trono. 1996. In vivo gene delivery and    stable transduction of nondividing cells by a lentiviral vector.    Science 272:263-267.-   34. O'Doherty, U., W. J. Swiggard, and M. H. Malim. 2000. Human    immunodeficiency virus type 1 spinoculation enhances infection    through virus binding. J. Virol. 74:10074-10080.-   35. Pacchia, A. L., M. E. Adelson, M. Kaul, Y. Ron, and J. P.    Dougherty. 2001. An inducible packaging cell system for safe,    efficient lentiviral vector production in the absence of HIV-1    accessory proteins. Virology 282:77-86.-   36. Perelson, A. S., P. Essunger, Y. Cao, M. Vesanen, A. Hurley, K.    Saksela, M. Markowitz, and D. D. Ho. 1997. Decay characteristics of    HIV-1-infected compartments during combination therapy. Nature    387:188-191.-   37. Prins, J. M., S. Jurriaans, R. M. van Praag, H. Blaak, R. van    Rij, P. T. Schellekens, I. J. ten Berge, S. L. Yong, C. H.    Fox, M. T. Roos, F. de Wolf, J. Goudsmit, H. Schuitemaker, and J. M.    Lange. 1999. Immuno-activation with anti-CD3 and recombinant human    IL-2 in HIV-1-infected patients on potent antiretroviral therapy.    AIDS 13:2405-2410.-   38. Purcell, D. F. and M. A. Martin. 1993. Alternative splicing of    human immunodeficiency virus type 1 mRNA modulates viral protein    expression, replication, and infectivity. J. Virol. 67:6365-6378.-   39. Roberts, B. D., G. Fang, and S. T. Butera. 1997. Influence of    cell cycle on HIV-1 expression differs among various models of    chronic infection. Arch. Virol. 142:1087-1099.-   40. Schwartz, S., B. K. Felber, D. M. Benko, E. M. Fenyo, and G. N.    Pavlakis. 1990. Cloning and functional analysis of multiply spliced    mRNA species of human immunodeficiency virus type 1. J Virol    64:2519-2529.-   41. Stellbrink, H. J., F. T. Hufert, K. Tenner-Racz, J. Lauer, C.    Schneider, H. Albrecht, P. Racz, and J. van Lunzen. 1998. Kinetics    of productive and latent HIV infection in lymphatic tissue and    peripheral blood during triple-drug combination therapy with or    without additional interleukin-2. Antivir. Ther. 3:209-214.-   42. Wang, F. X., Y. Xu, J. Sullivan, E. Souder, E. G. Argyris, E. A.    Acheampong, J. Fisher, M. Sierra, M. M. Thomson, R. Najera, I.    Frank, J. Kulkosky, R. J. Pomerantz, and G. Nunnari. 2005. IL-7 is a    potent and proviral strain-specific inducer of latent HIV-1 cellular    reservoirs of infected individuals on virally suppressive HAART. J.    Clin. Invest 115:128-137.-   43. Williams, S. A., L. F. Chen, H. Kwon, D. Fenard, D. Bisgrove, E.    Verdin, and W. C. Greene. 2004. Prostratin antagonizes HIV latency    by activating NF-kappaB. J. Biol. Chem. 279:42008-42017.-   44. Winslow, B. J., R. J. Pomerantz, O. Bagasra, and D. Trono. 1993.    HIV-1 latency due to the site of proviral integration. Virology    196:849-854.-   45. Wong, J. K., M. Hezareh, H. F. Gunthard, D. V. Havlir, C. C.    Ignacio, C. A. Spina, and D. D. Richman. 1997. Recovery of    replication-competent HIV despite prolonged suppression of plasma    viremia. Science 278:1291-1295.-   46. Yang, T. T., P. Sinai, P. A. Kitts, and S. R. Kain. 1997.    Quantification of gene expression with a secreted alkaline    phosphatase reporter system. Biotechniques 23:1110-1114.-   47. Zhang, J. and C. M. Sapp. 1999. Recombination between two    identical sequences within the same retroviral RNA molecule. J.    Virol. 73:5912-5917.-   48. Zhang, J. H., T. D. Chung, and K. R. Oldenburg. 1999. A Simple    Statistical Parameter for Use in Evaluation and Validation of High    Throughput Screening Assays. J. Biomol. Screen. 4:67-73.

1. An in vitro, cell-based method of identifying compounds capable ofactivating latent HIV-1 comprising: Providing an isolated SUPT1 cellline comprising a latent, HIV-1-derived provirus vector comprising: Anin-frame insertion of a secretable alkaline phosphatase (seap) gene openreading frame in place of the HIV-1 env gene start codon, aninactivation of the vpu gene start codon, a deletion of about 2500 basepairs of the HIV-1 pol gene, and an insertion of an egfp coding sequence10 base pairs upstream from the nef gene start codon, wherein uponactivation, the vector expresses the seap, a secretable marker for lateviral gene expression, or the egfp, a marker for early viral geneexpression; providing a candidate compound and a positive controlcompound for late viral gene activation, wherein the positive controlcompound is selected from tumor necrosis factor-α, phorbol 12-myristate13-acetate or valproic acid; combining the cell line with the candidatecompound and combining the cell line with the positive control compound;monitoring for seap expression in the presence of the candidate compoundas compared to seap expression in the absence of the candidate compoundand monitoring for seap expression in the presence of the positivecontrol to determine if the compound is capable of activating the vectorlate viral expression genes; monitoring for egfp expression in thepresence of the candidate compound as compared to egfp expression in theabsence of the candidate compound and monitoring for egfp expression inthe presence of the positive control to determine if the compound iscapable of activating the vector early viral expression genes; andwherein detecting increased seap expression by monitoring an amount ofseap expression that is greater in the presence of a candidate compoundthan in the absence of a candidate compound and/or detecting increasedegfp expression by monitoring an amount of egfp expression that isgreater in the presence of a candidate compound than in the absence of acandidate compound; and wherein detecting increased seap expression bymonitoring an amount of seap expression that is greater in the presenceof the positive control compound than in the absence of a the positivecontrol compound and detecting increased egfp expression by monitoringan amount of egfp expression that is greater in the presence of thepositive control compound than in the absence of the positive controlcompound, identifies a compound capable of activating latent HIV-1. 2.The method of claim 1, wherein the method further comprises detectingfluorescence by fluorescent microscopy, flow cytometry or combinationsthereof.
 3. The method of claim 1, wherein the provirus contains anintact HIV-1 Gag gene.
 4. The method of claim 3, further comprisingdetecting HIV-1 Gag expression.